Summary The goal of this project is to elucidate the biochemical basis of the functions of human replication protein A (RPA) in DNA damage responses, and to better understand the damage-induced mutagenesis and carcinogenesis. DNA damage has been widely implicated in human cancer induction. Human RPA, the major single-stranded DNA binding protein, is essential for all DNA metabolic pathways such as DNA replication, DNA repair, recombination and DNA damage checkpoints. Given that RPA undergoes hyperphosphorylation in cells in response to DNA damage, it is hypothesized that the protein may play a modulatory role in cellular DNA damage responses mediated by the hyperphosphorylation at the N- terminus of its RPA32 subunit. How the hyperphosphorylation affect the cellular activities of RPA in terms of biochemical mechanism remains unknown. In this study, using an array of rigorous biochemical methods we will: (1) identify domain-domain interactions and structural characteristic of RPA upon DNA damage-induced hyperphosphorylation;(2) characterize the effects of hyperphosphorylation on RPA interaction with DNA intermediates of biological significance;and (3) determine binding affinity and thermodynamics of the hyperphosphorylation-induced domain-domain interaction of RPA. The proposed study is highly relevant to cancer biology as human defects in DNA damage responses lead to genome instability and thus a series of diseases with high potential to evolve cancer. This Academic Research Enhancement Award will allow undergraduate and graduate students to gain biomedical research experience in a medical school environment that will prepare them for careers in scientific research. Specifically, the proposed study will address an important question regarding the biochemical basis of RPA hyperphosphorylation in cellular DNA damage responses. This project is highly relevant to cancer biology as DNA damage is the major cause to human cancer.